The prevalence of hemoglobin Tacoma in Finland detected by HbA1c capillary electrophoresis.

Previous studies have identified occasional cases of heterozygous Hb Tacoma in areas that have attracted Finnish immigrants, especially in Sweden and North America, but large studies of this slightly unstable beta variant in vitro have not been carried out. Here we determined the prevalence of hemoglobin variants across Finland. A total of 5059 samples from 11 different hospital districts were analyzed using HbA1c capillary electrophoresis and reviewed for atypical profiles (HbA1c, Capillarys 3 Tera, Sebia). 38 heterozygous Hb Tacoma cases were found (0.75%). The prevalence was highest in South Ostrobothnia (2.0%), located in western Finland, and second highest in the neighboring provinces (1.0-1.4%), but only two districts were devoid of variants. Heterozygous Hb Tacoma was confirmed by genetic testing (NM_000518.5(HBB):c.93G > T (p.Arg31Ser)). In addition, five other variants were found, suggestive of HbE, Hb Helsinki (two cases) and an alpha variant, as interpreted from the electropherograms. The fifth variant, belonging to the South Ostrobothnian cohort, remained unknown at the time of the initial analyses, but was later interpreted as homozygous Hb Tacoma and confirmed by hemoglobin fraction analysis (Hemoglobin(E), Capillarys 3 Tera). In a subsequent retrospective study of the electropherograms of routine HbA1c diagnostics, altogether nine homozygous Hb Tacoma cases were identified in South Ostrobothnia. While heterozygous Hb Tacoma is usually an incidental finding, it interferes with several HbA1c assays. The present study is the first demonstration of homozygous Hb Tacoma. The clinical presentations of homozygous Hb Tacoma are not known and need to be addressed in future studies.

Conformational changes of apoB-100 in SMase-modified LDL mediate formation of large aggregates at acidic pH.

During atherogenesis, the extracellular pH of atherosclerotic lesions decreases. Here, we examined the effect of low, but physiologically plausible pH on aggregation of modified LDL, one of the key processes in atherogenesis. LDL was treated with SMase, and aggregation of the SMase-treated LDL was followed at pH 5.5-7.5. The lower the pH, the more extensive was the aggregation of identically prelipolyzed LDL particles. At pH 5.5-6.0, the aggregates were much larger (size >1 µm) than those formed at neutral pH (100-200 nm). SMase treatment was found to lead to a dramatic decrease in α-helix and concomitant increase in ß-sheet structures of apoB-100. Particle aggregation was caused by interactions between newly exposed segments of apoB-100. LDL-derived lipid microemulsions lacking apoB-100 failed to form large aggregates. SMase-induced LDL aggregation could be blocked by lowering the incubation temperature to 15°C, which also inhibited the changes in the conformation of apoB-100, by proteolytic degradation of apoB-100 after SMase-treatment, and by HDL particles. Taken together, sphingomyelin hydrolysis induces exposure of protease-sensitive sites of apoB-100, whose interactions govern subsequent particle aggregation. The supersized LDL aggregates may contribute to the retention of LDL lipids in acidic areas of atherosclerosis-susceptible sites in the arterial intima.

Angiotensin II increases expression and secretion of cathepsin F in cultured human monocyte-derived macrophages: an angiotensin II type 2 receptor-mediated effect.

Increasing evidence suggests that cathepsins and angiotensin II (AngII) participate in atherosclerosis, particularly in remodeling of the extracellular matrix of the inflamed arterial intima. Here, we show that AngII induces mRNA expression of cathepsin F, a member of the cysteine protease family, in human monocyte-derived macrophages. AngII did not affect the amount of intracellular cathepsin F protein, but significantly enhanced its secretion by the treated cells. The stimulatory effect of AngII was mediated by the AngII type 2 (AT(2)) receptor, as demonstrated by the ability of the AT(2)-receptor antagonist PD123319 to block the AngII-induced increase in cathepsin F secretion. Our present data demonstrate a novel proatherogenic role for AngII, namely its ability to enhance secretion of lysosomal cathepsin F by monocyte-derived macrophages.

Miniaturization of asymmetrical flow field-flow fractionation and application to studies on lipoprotein aggregation and fusion.

Asymmetrical flow field-flow fractionation (AsFlFFF), a technique that provides direct measurement of particle size and diffusion coefficient, is converted into miniaturized scale. In comparison with conventional AsFlFFF, the separation of proteins in miniaturized AsFlFFF is achieved within shorter time periods, with smaller sample amounts, and with lower mobile phase consumption. Minimization of the overloading and optimization of the separation efficiency are prerequisites to good results. Miniaturized AsFlFFF is applied to the measurement of particle sizes of high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL). The average hydrodynamic diameters at pH 7.4 in 8.5mM phosphate buffer containing 1mM EDTA and 150 mM NaCl are 8.6+/-0.5, 11.2+/-0.2, 22.1+/-0.7, and 48.9+/-7.5 nm for subgroups HDL3, HDL2, LDL, and VLDL, respectively. In addition, the effect of different factors on the aggregation and fusion of LDL particles is studied. LDL particle sizes are unaffected by the addition of up to 300 mM NaCl and by an increase of the carrier solution pH from 3.2 to 7.4, but treatment of LDL with alpha-chymotrypsin, sphingomyelinase, or copper sulfate leads to the formation of aggregated and fused LDL particles.

Decrease in pH strongly enhances binding of native, proteolyzed, lipolyzed, and oxidized low density lipoprotein particles to human aortic proteoglycans.

Binding of low density lipoprotein (LDL) to proteoglycans and modification of LDL are key processes in atherogenesis. Recently, it has been demonstrated that during atherogenesis the extracellular pH of atherosclerotic lesions decreases. We have examined the effect of the decreased pH on the binding of LDL to human aortic proteoglycans. The binding of native, oxidized, proteolyzed (alpha-chymotrypsin-treated), or lipolyzed (sphingomyelinase- or phospholipase A(2)-treated) LDL particles to proteoglycans were measured in microtiter well assays at pH 5.5-7.5. We found that the lower the pH, the higher the amount of binding of LDL to proteoglycans. At the lowest pH tested (pH 5.5), the amounts of proteoglycan-bound native, proteolyzed, sphingomyelinase-, and phospholipase A(2)-treated LDL were 20-, 23-, 30-, and 37-fold higher, respectively, than at pH 7.5. Interestingly, although oxidized LDL failed to bind to proteoglycans at neutral pH, there was significant binding at acidic pH. Binding of native and modified LDL to proteoglycans at pH 5.5 was blocked by 1 m NaCl, indicating that at neutral pH LDL binds to proteoglycans via ionic interactions. Inhibition of this binding by acetylation and cyclohexanedione treatment of LDL showed that the positively charged amino acids of apolipoprotein B-100, lysine, and arginine, respectively, mediated the ionic interaction. Taken together, our results suggest that in areas of atherosclerotic arterial intima where the extracellular pH decreases, retention of LDL by proteoglycans is enhanced, leading to extracellular accumulation of LDL and progression of the disease.

Radionuclide imaging of tumor xenografts in mice using a gelatinase-targeting peptide.

Tumors express MMP-2 and MMP-9 gelatinases, which are involved in the formation of tumor vasculature. This suggests that a tumor and its surrounding neovasculature can be visualized by a sensitive gelatinase recognition method. We have studied tumor radioimaging using a gelatinase inhibitory peptide CTTHWGFTLC (CTT), which in a mouse model targets the tumor site following an intravenous injection. We determined a solution NMR structure of CTT and its retro-inversion peptide, and prepared 125I and 99mTc-labelled CTT peptide derivatives. Radiolabelled CTT inhibited gelatinases in vitro, and homed to a tumor xenograft in mice. In normal mice, CTT was instead rapidly cleared from the circulation mainly through the kidney and, after 24 h, no significant radioactivity was accumulated in healthy tissues. Gamma camera imaging of a primary tumor in live mice was obtained with double-labelled liposomes, which were coated with 99mTc-CTT and encapsulated with 125I albumin. CTT also targeted liposomes to the lungs of tumor-bearing mice, which may indicate the existence of non-visible lung micrometastases. Our studies suggest that selective gelatinase-targeting compounds could be useful in the early detection and imaging of primary tumors and metastases.

Cysteine protease cathepsin F is expressed in human atherosclerotic lesions, is secreted by cultured macrophages, and modifies low density lipoprotein particles in vitro.

During atherogenesis, low density lipoprotein (LDL) particles in the arterial intima become modified and fuse to form extracellular lipid droplets. Proteolytic modification of apolipoprotein (apo) B-100 may be one mechanism of droplet formation from LDL. Here we studied whether the newly described acid protease cathepsin F can generate LDL-derived lipid droplets in vitro. Treatment of LDL particles with human recombinant cathepsin F led to extensive degradation of apoB-100, which, as determined by rate zonal flotation, electron microscopy, and NMR spectroscopy, triggered both aggregation and fusion of the LDL particles. Two other acid cysteine proteases, cathepsins S and K, which have been shown to be present in the arterial intima, were also capable of degrading apoB-100, albeit less efficiently. Cathepsin F treatment resulted also in enhanced retention of LDL to human arterial proteoglycans in vitro. Cultured monocyte-derived macrophages were found to secrete active cathepsin F. In addition, similarly with cathepsins S and K, cathepsin F was found to be localized mainly within the macrophage-rich areas of the human coronary atherosclerotic plaques. These results suggest that proteolytic modification of LDL by cathepsin F may be one mechanism leading to the extracellular accumulation of LDL-derived lipid droplets within the proteoglycan-rich extracellular matrix of the arterial intima during atherogenesis.